Blotting Immunoblotting

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

Key Words Western blotting immunoblotting electrophoresis 2D electrophoresis immunoaffinity identification immunoblotting techniques. 

The immunoassays (Dot-blot, immunoblotting, ELISA) are often applied in combinations, for example, to characterize immunoreactive (allergenic) properties of fractions obtained via mechanical, electrophoretic (SDS-PAGE, 2-D), or... 

Immunochemical properties Binding assays, ELISA, western-blot (immunoblotting methods)... 

Grinnell, E, Ho, C. H., and Wysodd, A. 1992. Degradation of fibronectin and vitronectin in chronic wound fluid analysis by ceU blotting, immunoblotting, and cell adhesion assays. Journal of Investigative Dermatology, 98,410-416. 

Fig. 2. Tissue-print immunoblots of cross sections from okrafruitpods. Ponceau S protein staining of blots before immunodetection reveals the anatomical detail of sections subsequently probed with control serum (A) or with Phaseolus vulgaris IAA-peptide antibody (B). Arrows indicate the position of seeds remaining within the cross-sectioned pods.

Antibodies may also be used to determine the presence or identity of soluble antigens by a process known generally as immunoblotting. In a technique known as dot blotting , soluble antigens are applied to a nitrocellulose membrane in the form of dots , antibody is then applied to the membrane, the membrane is washed to remove unbound antibody, and the presence or absence of the antigen is determined by similar means to that employed during immunohistochemistry. 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

Proteins bound to the surfaces of synthetic membranes retain their antigenicity and are accessible to antibody probes. The most common membrane-based immunoassay technique is called immunoblotting or, more popularly, Western blotting. In Western blotting, proteins are transferred from an electrophoresis gel to a... 

Figure 6.5. Immunoblot of the urease large subunit. Extracts of H. pylori wild type (WT) and a hypA mutant from cells grown without nickel supplementation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by blotting with an anti-urease large-subunit antiserum. Urease activity was 58pmolmin mg for the wild type and Opmolmin" mg for the hypA mutant.

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

Western blots Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen. 

Western blotting—used to identify a specific protein or group of proteins by immunoblotting (detection by antibodies). 

---